Tomato plants with improved disease resistance

ABSTRACT

The present disclosure provides tomato plants exhibiting resistance to tomato yellow leaf curl virus (TYLCV) and lacking unfavorable linked traits such as necrosis. Such plants may comprise novel introgressed genomic regions associated with disease resistance from  S. chilense . In certain aspects, compositions, including novel polymorphic markers and methods for producing, breeding, identifying, and selecting plants or germplasm with a disease resistance phenotype are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/064,375, filed Oct. 15, 2014, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture and morespecifically to methods and compositions for producing tomato plantsexhibiting disease resistance with reduced plant necrosis.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB019US_ST25,” which is 11.1 kilobytes as measured in MicrosoftWindows operating system and was created on Oct. 13, 2015, is filedelectronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

Disease resistance is an important trait in agriculture, particularlyfor the production of food crops. Although disease resistance alleleshave been identified in uncultivated tomato lines, efforts to introducethese alleles into cultivated lines are hindered by the introduction ofdeleterious traits together with the resistance alleles. The use ofmarker-assisted selection (MAS) in plant breeding methods has made itpossible to select plants based on genetic markers linked to traits ofinterest. However, accurate markers for identifying or trackingdesirable traits in plants are frequently unavailable even if a geneassociated with the trait has been characterized. These difficulties arefurther complicated by factors such as polygenic or quantitativeinheritance, and an often incomplete understanding of the geneticbackground underlying expression of a desired phenotype. Therefore, inthe absence of accurate and validated markers for use in MAS, it may notbe feasible to produce new plant lines exhibiting certain diseaseresistant phenotypes.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a tomato plant of a cultivatedtomato plant variety comprising a recombinant introgression from Solanumchilense on chromosome 6, wherein said recombinant introgressioncomprises a first allele conferring improved resistance to tomato yellowleaf curl virus relative to a plant lacking said first allele, andwherein said recombinant introgression lacks a second allele geneticallylinked to said first allele and conferring necrosis. In certainembodiments, the recombinant introgression from Solanum chilense islocated between approximately 30.20 Mbp and 30.88 Mbp on chromosome 6,or is located between approximately 30.69 Mbp and 30.88 Mbp onchromosome 6, or is located between approximately 30.71 Mbp and 30.88Mbp on chromosome 6, or is located between approximately 30.32 Mbp and30.71 Mbp on chromosome 6, or is located between approximately 30.88 Mbpand 31.80 Mbp on chromosome 6.

In other embodiments, the plant comprises a Solanum chilense allele atlocus NSLYC008383071 (SEQ ID NO:1) and lacks a Solanum chilense alleleat locus NSLYC005134429 (SEQ ID NO:3) or the plant comprises a Solanumchilense allele at locus NSLYC008383071 (SEQ ID NO:1) and lacks aSolanum chilense allele at locus NSLYC009077970 (SEQ ID NO:2), or theplant comprises S. chilense donor DNA within a genomic segment flankedby NSLYC005134429 (SEQ ID NO:3) and NSLYC008383071 (SEQ ID NO:1), or theplant comprises S. chilense donor DNA within a genomic segment flankedby NSLYC009077970 (SEQ ID NO:2) and NSLYC008383071 (SEQ ID NO:1), or theplant comprises S. chilense donor DNA within a genomic segment flankedby NSLYC008382908 and NSLYC008383071 (SEQ ID NO:1), or the plantcomprises S. chilense donor DNA within a genomic segment flanked byNSLYC009418370 (SEQ ID NO:4) and NL231193. In another embodiment, theinvention provides a plant part of such a plant, including a cell, aseed, a root, a stem, a leaf, a fruit, a flower, or pollen.

In another aspect, the invention provides a method for producing atomato plant with improved resistance to tomato yellow leaf curl viruscomprising: a) crossing the tomato plant of claim 1 with itself or witha second tomato plant of a different genotype to produce one or moreprogeny plants; and b) selecting a progeny plant comprising saidrecombinant introgression. In one embodiment, selecting the progenyplant comprises identifying a progeny plant that (1) comprises a Solanumchilense allele at a locus genetically linked to said first alleleand/or lacks an allele present at the corresponding locus in the tomatoplant, and (2) lacks a Solanum chilense allele at a locus geneticallylinked to said second allele that confers necrosis, and/or comprises anallele present at the corresponding locus from in the tomato plant. Inanother embodiment, selecting said progeny plant comprisesmarker-assisted selection (MAS). In a further embodiment,marker-assisted selection (MAS) comprises detecting at least one alleleat a locus selected from the group consisting of NSLYC005134429 (SEQ IDNO:3), NSLYC009077970 (SEQ ID NO:2), NSLYC009418370 (SEQ ID NO:4),NSLYC008383071 (SEQ ID NO:1), NSLYC008382908, and NL231193. In stillfurther embodiments, the progeny plant is an F2-F6 progeny plant, orproducing the progeny plant comprises backcrossing, such as from 2-7generations of backcrossing.

In another aspect, the invention provides a method for obtaining atomato plant exhibiting improved resistance to tomato yellow leaf curlvirus comprising: a) obtaining a tomato plant heterozygous for a firstallele that confers resistance to tomato yellow leaf curl virus and thatis genetically linked in the plant to a second allele from Solanumchilense that confers necrosis; (b) obtaining progeny of the plant; and(c) selecting at least a first progeny plant in which recombination hasoccurred such that the progeny comprises said first allele that confersresistance to tomato yellow leaf curl virus but not said second allelethat confers necrosis; wherein selecting said first progeny plantcomprises detecting at least one allele at a locus selected from thegroup consisting of NSLYC005134429 (SEQ ID NO:3), NSLYC009077970 (SEQ IDNO:2), NSLYC009418370 (SEQ ID NO:4), NSLYC008383071 (SEQ ID NO:1),NSLYC008382908, and NL231193. In one embodiment, the progeny plant is anF2-F6 progeny plant, or producing the progeny plant comprisesbackcrossing, such as from 2-7 generations of backcrossing. In anotherembodiment, the invention provides a plant produced by such a method, ora part of such a plant, selected from the group consisting of a cell, aseed, a root, a stem, a leaf, a fruit, a flower, and pollen.

In another aspect, the invention provides a tomato plant of a cultivatedtomato plant variety comprising a TT allele at marker NSLYC008374675(SEQ ID NO:5), and a GG allele at marker NSLYC008375578 (SEQ ID NO:6),wherein said alleles confer a lack of or resistance to necrosis. In oneembodiment, the method further comprises: d) selecting against alleleson chromosome 2 associated with necrosis; wherein said selecting againstcomprises selecting against plants comprising a CC allele at markerNSLYC008374675 (SEQ ID NO:5), or an AA allele at marker NSLYC008375578(SEQ ID NO:6), wherein the presence of said alleles confers necrosis.

In another aspect, the invention provides a method for obtaining atomato plant exhibiting resistance to necrosis comprising: a) obtaininga tomato plant heterozygous for a first allele that confers resistanceto necrosis; (b) obtaining progeny of the plant; and (c) selecting atleast a first progeny plant in which recombination has occurred suchthat the progeny comprises said first allele that confers resistance tonecrosis; wherein selecting said first progeny plant comprises detectingat least one allele at a locus selected from the group consisting ofmarker NSLYC008374675 (SEQ ID NO:5) and marker NSLYC008375578 (SEQ IDNO:6), wherein said alleles confer a lack of or resistance to necrosis.

In another aspect, the invention provides a method for obtaining atomato plant exhibiting improved resistance to tomato yellow leaf curlvirus comprising: (a) obtaining a tomato plant heterozygous for a firstallele that confers resistance to tomato yellow leaf curl virus and thatis genetically linked in the plant to a second allele from Solanumchilense that confers necrosis; (b) obtaining progeny of the plant; and(c) selecting at least a first progeny plant in which recombination hasoccurred such that the progeny comprises said first allele that confersresistance to tomato yellow leaf curl virus but not said second allelethat confers necrosis. In accordance with the invention, selecting aprogeny plant may comprise selecting a plant that comprises an allelethat confers resistance to tomato yellow leaf curl virus from Solanumchilense chromosome 6, and lacks a second locus linked theretoconferring one or more of necrosis, reduced fruit set, or reduced fruitsize, relative to a plant that lacks the second locus. In one embodimentof the method, selecting said first progeny plant comprises selecting aprogeny wherein recombination has occurred between locus NSLYC009418370(SEQ ID NO:4) and locus NL231193. In a specific embodiment, the methodcomprises detecting at least one allele at a locus selected from thegroup consisting of NSLYC008383071 (SEQ ID NO:1), NSLYC009418370 (SEQ IDNO:4), NSLYC008382908, and NL231193. In another embodiment, the methodcomprises (d) selecting a further progeny plant from among the plantsselected in step (c); wherein selecting said further progeny plantcomprises detecting at least one allele at a locus selected from thegroup consisting of NSLYC009418370 (SEQ ID NO:4) and NL231193. In yetanother embodiment, the invention provides a plant produced by any ofthe foregoing methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows a comparison of recombinants in the Mi (nematode)-Ty(geminivirus) region of chromosome 6. Columns from left to right are:genetic sources, 22 marker assays including marker for the nematodetrait (1), and three Ty-1 markers for the geminivirus trait (2-4),followed by phenotyping results from geminivirus agroinoculation, fieldresistance, and an assessment of leaf necrosis, which is a manifestationof genetic drag from the Ty-1 introgression. Genotypes are indicated byshading as shown in the figure legend. “nd” indicates no data.

FIG. 2: Shows an LOD plot derived from genotypic and phenotypic data inan F2 population comprising a Ty-1 introgression from S. chilense.

FIG. 3: Shows the average necrosis rating for several F3 linescomprising Ty-1 introgressions, together with the genotype at severalmarker loci. “esc” refers to DNA originating from S. esculentum at thegiven marker locus, and “chi” refers to DNA originating from S. chilenseat the given marker locus.

FIG. 4: Shows the average necrosis score for several of the plant linesshown in FIG. 3 based on the location of the break point between donorS. chilense DNA and S. esculentum DNA determined using the markers shownin FIG. 3.

FIG. 5: Shows the average necrosis rating for several plant lines,together with genotypes at two marker loci on chromosome 2 associatedwith necrosis as shown in FIG. 2.

FIG. 6: Shows the genotype of a set of F4 recombinant lines at severalmarker loci in the approximate region of Ty-1 (between 20 and 34 Mbp ofchromosome 6). The highly correlated TaqMan assay based on a SNP isNSLYC008426102. A gray SNP means identical to SNP present in susceptibleparents (FIR-16-1078 and FDR-16-0197) and a white SNP means identical toSNP present in resistant parents (FIR-AY06074 and FDR-16-2133). In thesecond column, the TYLCV test scores are shown.

FIG. 7: Shows the genotype of a set of recombinant lines at severalmarker loci in the approximate region of Ty-1 (between 30.3 Mbp and 31.8Mbp of chromosome 6). Gray indicates a donor S. chilense allele andwhite indicates a recipient S. esculentum allele.

FIG. 8: Shows the results of performance testing for fruit set (totalnumber of mature fruit per number of plants).

FIG. 9: Shows the results of performance testing for fruit set (totalharvest weight in kg per number of fruit).

FIG. 10: Shows a depiction of the introgression (approximately 260 kb).

DETAILED DESCRIPTION

Tomato yellow leaf curl virus (TYLCV) is a plant pathogenic virus whichis responsible for severe yield loss in tomato plants. Several wildtomato species are known to exhibit resistance to TYLCV, and intensiveefforts have been made to introgress TYLCV resistance alleles from thesespecies into cultivated tomato lines. However, these efforts have beenhampered because resistance alleles from wild species are accompanied byundesirable agronomic traits such as necrosis. Yield loss due to TYLCVin tomato plants remains a significant problem.

For the first time, the invention provides novel introgressions ofdisease resistance alleles from Solanum chilense into cultivated orelite tomato lines, resulting in tomato plants exhibiting high levels ofresistance to TYLCV without the deleterious necrosis previouslyassociated with Ty1 introgressions from wild species. In a specificembodiment, the present invention may be used in the Lycopersiconesculentum (aka Solanum lycopersicum) species of tomato. The inventiontherefore represents a significant advance in the art. By furtherproviding novel, accurate markers for tracking the introgressed alleleswithout associated necrosis during plant breeding, the invention permitsintrogression of the disease resistance into any desired tomatogenotype.

Resistance to TYLCV has conventionally been obtained throughintrogressions of the Ty-1 locus from S. chilense. However, Ty-1introgressions are typically large, and plants comprising theseintrogressions exhibit agronomically unacceptable traits includingnecrosis. Efforts to reduce the incidence or severity of necrosis inplants comprising Ty-1 introgressions have been unsuccessful in part dueto suppressed recombination in the centromeric region of chromosome 6near the Ty-1 locus. In addition, several genetic regions surroundingthe Ty-1 locus are inverted in wild tomato lines compared withcultivated lines, further interfering with recombination. Introgressionof Ty-1 alleles from S. chilense is even further complicated by a lackof existing markers and assays that accurately correlate genotype withresistance and reduced necrosis over a variety of tomato lines.

Despite the many obstacles to the successful introgression of Ty-1resistance alleles from S. chilense into cultivated tomato lines, thepresent inventors were surprisingly able to produce novel introgressionsfrom S. chilense which confer resistance to TYLCV without thedeleterious traits previously associated with Ty-1 introgressions. Insome embodiments, the invention provides plants comprising donor S.chilense DNA at marker NSLYC008383071 (SEQ ID NO:1) and recipient DNA atmarker NSLYC009077970 (SEQ ID NO:2). In other embodiments, the inventionprovides plants comprising donor S. chilense DNA at markerNSLYC008383071 (SEQ ID NO:1) and recipient DNA at marker NSLYC005134429(SEQ ID NO:3). In other embodiments, the invention provides plantscomprising S. chilense donor DNA within a genomic segment flanked byNSLYC009077970 (SEQ ID NO:2) and NSLYC008383071 (SEQ ID NO:1) or withina genomic segment flanked by NSLYC005134429 (SEQ ID NO:3) andNSLYC008383071 (SEQ ID NO:1).

The present invention further provides novel trait-linked markers whichcan be used to produce plants comprising novel recombined introgressionsin the Ty-1 region of chromosome 6 conferring TYLCV resistance withoutnecrosis. In particular embodiments, the invention provides the markersshown in Tables 1, 4, 6, and 7. Other embodiments of the inventionprovide novel markers NSLYC008383071 (SEQ ID NO:1), NSLYC009418370 (SEQID NO:4), NSLYC009077970 (SEQ ID NO:2), and NSLYC005134429 (SEQ ID NO:3)which have been shown to be genetically linked to TYLCV resistance inplants.

The invention further provides Ty-1 introgressions which can be deployedhomozygously without detrimental necrosis or other unacceptable traits.Ty-1 alleles have conventionally been deployed heterozygously in aneffort to reduce the severity of the necrotic symptoms exhibited byplants comprising Ty-1 introgressions. However, contrary to the beliefin the field that heterozygous deployment of Ty-1 alleles derived fromS. chilense in an elite line was necessary to avoid negative traits suchas necrosis, the present invention provides Ty-1 resistance alleleswhich can be deployed homozygously without unacceptable levels ofnecrosis in plants. The novel, reduced introgressions of the presentinvention therefore provide significant advantages over existingtechnology.

The invention further identifies a novel QTL conferring resistance tonecrosis on tomato chromosome 2, as well as nucleic acid sequences andgenetic markers associated with the QTL. The use of the novel markersprovided herein for selection of plants having favorable alleles withinor genetically linked to this locus allows for the development of plantswith reduced or absent necrotic symptoms. In some embodiments, theinvention therefore provides methods of producing plants havingdecreased or absent necrotic symptoms by selecting or breeding plantshaving favorable alleles at the markers on chromosome 2 disclosedherein. In certain embodiments, the invention provides plants having aTT allele within marker NSLYC008374675 (SEQ ID NO:5) and plants having aGG allele within marker NSLYC008375578 (SEQ ID NO:6) which exhibitdecreased necrosis compared with plants not comprising these alleles.

The invention further provides novel markers and assays that allow theaccurate identification and tracking of the genomic regions providedherein during plant breeding. Because genetically diverse tomato linescan be difficult to cross due in part to suppressed recombination in theTy-1 region, the introduction of TYLCV resistance alleles withoutassociated necrosis from S. chilense into elite tomato lines orcultivated lines using conventional breeding methods would requireprohibitively large segregating populations for progeny screens with anuncertain outcome. Marker-assisted selection (MAS) is therefore animprovement in the effective introgression of wild tomato alleles intoelite cultivars. However, previously known markers for TYLCV resistancehave not allowed selection of TYLCV resistance without associatednecrosis. In contrast, the present invention enables MAS of Ty1 withoutassociated necrosis by providing improved and validated markers fordetecting genotypes associated with disease resistance and reducednecrosis without the need to grow large populations of plants tomaturity in order to observe the phenotype.

I. Genomic Regions, Alleles, and Polymorphisms Associated with TYLCVResistance and Reduced Necrosis in Tomato Plants

The invention provides novel introgressions of one or more allelesassociated with disease resistance and reduced plant necrosis in tomatoplants, together with polymorphic nucleic acids and linked markers fortracking the introgressions during plant breeding.

TYLCV can infect tomato plants at any stage in the growth cycle and cancause severe reduction in yield and quality in a tomato crop. Intensiveefforts have therefore been made to identify effective sources of TYLCVresistance. However, previously known introgressions from wild specieshave been associated with plant necrosis. In particular, cultivatedtomato lines carrying previously known introgressions of TYLCVresistance genes exhibit necrosis of the leaves. Despite many years ofselective breeding in an effort to reduce the incidence of necrosis,these effects are still routinely observed in the field.

Wild tomato types exhibiting TYLCV resistance, for example S. chilense,are known in the art and may be used in accordance with certainembodiments of the invention. Other TYLCV resistance sources have alsobeen described and are known in the art (see, for example, Vidaysky etal., Phytopathology, 88(9):910-4, 1998; Hutton et al., HortScience,47(3):324-327, 2012; Ji et al., HortScience, 44(3):614-618, 2009).

Using the improved genetic markers and assays of the invention,Applicants were able to successfully identify a novel TYLCV resistanceregion from S. chilense associated with fewer deleterious traits whenintrogressed into a cultivated line. In certain embodiments, theinvention provides plants comprising donor S. chilense DNA at markerNSLYC008383071 (SEQ ID NO:1) and recipient DNA at marker NSLYC009077970(SEQ ID NO:2). In other embodiments, the invention provides plantscomprising donor S. chilense DNA at marker NSLYC008383071 (SEQ ID NO:1)and recipient DNA at marker NSLYC005134429 (SEQ ID NO:3). In otherembodiments, the invention provides plants comprising S. chilense donorDNA within a genomic segment flanked by NSLYC009077970 (SEQ ID NO:2) andNSLYC008383071 (SEQ ID NO:1) or a genomic segment flanked byNSLYC005134429 (SEQ ID NO:3) and NSLYC008383071 (SEQ ID NO:1).

The invention further identifies and provides genomic segments fromapproximately 30.20 Mbp (NSLYC005134429, SEQ ID NO:3) to 30.88 Mbp(NSLYC009418370, SEQ ID NO:4), from approximately 30.69 Mbp(NSLYC009077970, SEQ ID NO:2) to 30.88 Mbp (NSLYC009418370, SEQ IDNO:4), or from approximately 30.71 Mbp (NSLYC008383071, SEQ ID NO:1) to30.88 Mbp (NSLYC009418370, SEQ ID NO:4) on chromosome 6 associated withTYLCV resistance, but not associated with necrosis when introgressedinto cultivated tomato lines. In particular embodiments, the inventionprovides a plant comprising recipient DNA at one or more of markersNL0232061, NL0216350, NL0244835, NSLYC005134413, NSLYC005134429 (SEQ IDNO:3), and NSLYC008382908, and comprising S. chilense donor DNA at oneor more of markers NSLYC008373232, NSLYC009078368, NSLYC009077969,NSLYC009077970 (SEQ ID NO:2), NSLYC008383071 (SEQ ID NO:1),NSLYC009418370 (SEQ ID NO:4), NSLYC008426177, and NL231193, whichexhibits resistance to TYLCV and does not exhibit necrosis.

In another embodiment, the invention provides novel markers that may beused to identify a locus as described herein, such as the markers setforth in Tables 1, 4, 6, and 7. Other embodiments of the inventionprovide novel markers NSLYC008383071 (SEQ ID NO:1), NSLYC009418370 (SEQID NO:4), NSLYC009077970 (SEQ ID NO:2), and NSLYC005134429 (SEQ ID NO:3)which have been shown to be genetically linked to TYLCV resistance inplants.

II. Introgression of Genomic Regions Associated with Disease Resistance

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first genetic background toa second. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first genetic backgroundand both linked and unlinked markers characteristic of the secondgenetic background.

The present invention provides novel accurate markers for identifyingand tracking introgression of one or more of the genomic regions from S.chilense disclosed herein into cultivated lines. The invention furtherprovides markers for identifying and tracking the novel introgressionsdisclosed herein during plant breeding, including markers set forth inTables 1, 4, 6, and 7. Other embodiments of the invention provide novelmarkers NSLYC008383071 (SEQ ID NO:1), NSLYC009418370 (SEQ ID NO:4),NSLYC009077970 (SEQ ID NO:2), and NSLYC005134429 (SEQ ID NO:3) whichhave been shown to be genetically linked to TYLCV resistance in plants.

Markers within or linked to any of the genomic intervals of the presentinvention may be useful in a variety of breeding efforts that includeintrogression of genomic regions associated with disease resistance intoa desired genetic background. For example, a marker within 40 cM, 20 cM,15 cM, 10 cM, 5 cM, 2 cM, or 1 cM of a marker associated with diseaseresistance described herein can be used for marker-assistedintrogression of genomic regions associated with a disease tolerantphenotype.

Tomato plants comprising one or more introgressed regions associatedwith a desired phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or99% of the remaining genomic sequences carrying markers characteristicof the germplasm are also provided. Tomato plants comprising anintrogressed region comprising regions closely linked to or adjacent tothe genomic regions and markers provided herein and associated with adisease resistance phenotype are also provided.

III. Development of Disease Resistant Tomato Varieties

For most breeding objectives, commercial breeders work within germplasmthat is “cultivated,” “cultivated type” or “elite.” “Elite” varietiesrefer to varieties that have resulted from breeding and selection forsuperior agronomic performance, including yield and other selectedtraits. Similarly, “cultivated” varieties or “cultivars” are plants orgroups of plants selected for desirable characteristics for cultivationin agriculture and that can be maintained through propagation of thevariety. Elite varieties or cultivars are easier to breed because theygenerally perform well when evaluated for horticultural performance. Anumber of cultivated and elite tomato (S. lycopersicum or L. esculentum)types have been developed, which are agronomically superior andappropriate for commercial cultivation. However, the performanceadvantage a cultivated germplasm provides can be offset by a lack ofallelic diversity. Breeders generally accept this tradeoff becauseprogress is faster when working with cultivated material than whenbreeding with genetically diverse sources. Thus, in certain embodiments,the present invention provides novel introgressions of diseaseresistance alleles from Solanum chilense into an elite tomato line, orinto a cultivated tomato line, resulting in tomato plants exhibitinghigh levels of resistance to TYLCV without the deleterious necrosispreviously associated with Ty1 introgressions from wild species. Suchintrogressions may provide superior tomato plants that may possess anumber of agronomically elite traits, in addition to the novelintrogression of the present invention.

In contrast, when cultivated germplasm is crossed with non-cultivatedgermplasm, a breeder can gain access to novel alleles from thenon-cultivated type. However, this approach presents significantdifficulties due to fertility problems associated with crosses betweendiverse lines, and negative linkage drag from the non-cultivated parent.In tomato plants, non-cultivated types such as S. chilense can providealleles associated with disease resistance. However, thesenon-cultivated types may have poor horticultural qualities such asvulnerability to necrosis or fruit defects.

The process of introgressing desirable resistance genes fromnon-cultivated lines into elite cultivated lines while avoiding problemswith linkage drag or low heritability is a long and often arduousprocess. Success in deploying alleles derived from wild relativestherefore strongly depends on minimal or truncated introgressions thatlack detrimental effects and reliable marker assays that replacephenotypic screens. Success is further defined by simplifying geneticsfor key attributes to allow focus on genetic gain for quantitativetraits such as disease resistance. Moreover, the process ofintrogressing genomic regions from non-cultivated lines can be greatlyfacilitated by the availability of accurate markers for MAS.

One of skill in the art would therefore understand that the alleles,polymorphisms, and markers provided by the invention allow the trackingand introduction of any of the genomic regions identified herein intoany genetic background. In addition, the genomic regions associated withdisease resistance disclosed herein can be introgressed from onegenotype to another and tracked using MAS. Thus, Applicants' discoveryof accurate markers associated with disease resistance will facilitatethe development of tomato plants having beneficial phenotypes. Forexample, seed can be genotyped using the markers of the presentinvention in order to select for plants comprising desired genomicregions associated with disease resistance. Moreover, MAS allowsidentification of plants homozygous or heterozygous for a desiredintrogression.

Inter-species crosses can also result in suppressed recombination andplants with low fertility or fecundity. For example, suppressedrecombination has been observed for the tomato nematode resistance geneMi, the Mla and Mlg genes in barley, the Yr17 and Lr20 genes in wheat,the Run1 gene in grapevine, and the Rma gene in peanut. Meioticrecombination is essential for classical breeding because it enables thetransfer of favorable alleles across genetic backgrounds, the removal ofdeleterious genomic fragments, and pyramiding of traits that aregenetically linked. Therefore, in the absence of accurate markers,suppressed recombination forces breeders to enlarge segregatingpopulations for progeny screens.

Phenotypic evaluation of large populations is time-consuming,resource-intensive and not reproducible in every environment.Marker-assisted selection offers a feasible alternative. Molecularassays designed to detect unique polymorphisms, such as SNPs, areversatile. However, they may fail to discriminate alleles within andamong tomato species in a single assay. Structural rearrangements ofchromosomes such as deletions impair hybridization and extension ofsynthetically labeled oligonucleotides. In the case of duplicationevents, multiple copies are amplified in a single reaction withoutdistinction. The development and validation of accurate and highlypredictive markers are therefore essential for successful MAS breedingprograms.

IV. Molecular Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),simple sequence repeats (SSRs), simple sequence length polymorphisms(SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletionpolymorphisms (Indels), variable number tandem repeats (VNTRs), andrandom amplified polymorphic DNA (RAPD), isozymes, and other markersknown to those skilled in the art. Marker discovery and development incrop plants provides the initial framework for applications tomarker-assisted breeding activities (U.S. Patent Pub. Nos.:2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). Theresulting “genetic map” is the representation of the relative positionof characterized loci (polymorphic nucleic acid markers or any otherlocus for which alleles can be identified) to each other.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism (Orita et al. (1989) Genomics, 8(2), 271-278), denaturinggradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavagefragment length polymorphisms (Life Technologies, Inc., Gathersberg,Md.), but the widespread availability of DNA sequencing often makes iteasier to simply sequence amplified products directly. Once thepolymorphic sequence difference is known, rapid assays can be designedfor progeny testing, typically involving some version of PCRamplification of specific alleles (PASA; Sommer, et al. (1992)Biotechniques 12(1), 82-87), or PCR amplification of multiple specificalleles (PAMSA; Dutton and Sommer (1991) Biotechniques, 11(6),700-7002).

Polymorphic markers serve as useful tools for assaying plants fordetermining the degree of identity of lines or varieties (U.S. Pat. No.6,207,367). These markers form the basis for determining associationswith phenotypes and can be used to drive genetic gain. In certainembodiments of methods of the invention, polymorphic nucleic acids canbe used to detect in a tomato plant a genotype associated with diseaseresistance, identify a tomato plant with a genotype associated withdisease resistance, and to select a tomato plant with a genotypeassociated with disease resistance. In certain embodiments of methods ofthe invention, polymorphic nucleic acids can be used to produce a tomatoplant that comprises in its genome an introgressed locus associated withdisease resistance. In certain embodiments of the invention, polymorphicnucleic acids can be used to breed progeny tomato plants comprising alocus associated with disease resistance.

Genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e. for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with disease resistance in tomatoplants.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol.51:263-273; European Patent 50,424; European Patent 84,796; EuropeanPatent 258,017; European Patent 237,362; European Patent 201,184; U.S.Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No.4,683,194), using primer pairs that are capable of hybridizing to theproximal sequences that define a polymorphism in its double-strandedform. Methods for typing DNA based on mass spectrometry can also beused. Such methods are disclosed in U.S. Pat. Nos. 6,613,509 and6,503,710, and references found therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods, for example as disclosed in U.S. Pat. No. 5,800,944 wheresequence of interest is amplified and hybridized to probes followed byligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe has a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR, forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle, DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, a locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays.

DEFINITIONS

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which tomato plants canbe regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, an “elite” variety means any variety that has resultedfrom breeding and selection for superior agronomic performance includingyield and other selected traits. An “elite plant” refers to a plantbelonging to an elite variety and which possesses various desirabletraits. Numerous elite varieties are available and known to those ofskill in the art of tomato breeding. An “elite population” is anassortment of elite individuals or varieties that can be used torepresent the state of the art in terms of agronomically superiorgenotypes of a given crop species, such as tomato. Similarly, an “elitegermplasm” or elite strain of germplasm is an agronomically superiorgermplasm.

A “cultivated” variety or “cultivar” is a plant or group of plantsselected for desirable characteristics when grown in agriculture thatcan be maintained through propagation of the variety. A cultivatedvariety is not naturally occurring in the wild, and is instead theresult of human intervention to obtain desirable characteristics forcultivation.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can be achieved through plant breeding methods and/or bymolecular genetic methods. Such molecular genetic methods include, butare not limited to, various plant transformation techniques and/ormethods that provide for homologous recombination, non-homologousrecombination, site-specific recombination, and/or genomic modificationsthat provide for locus substitution or locus conversion.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

As used herein, the term “necrosis,” when used in the context of a plantor plant tissue, refers to tissue that turns brown and dies. As usedherein, “necrosis” may also include the incidence of chlorosis, wherechlorotic tissue is marked by the yellow or white discoloration ofnormally green tissue.

As used herein, “resistance locus” means a locus associated withresistance or tolerance to disease. For instance, a resistance locusaccording to the present invention may, in one embodiment, controlresistance or susceptibility for tomato yellow leaf curl virus (TYLCV).

As used herein, “resistance allele” means the nucleic acid sequenceassociated with resistance or tolerance to disease.

As used herein “resistance” or “improved resistance” in a plant todisease conditions is an indication that the plant is more able toreduce disease burden than a non-resistant or less resistant plant.Resistance is a relative term, indicating that a “resistant” plant ismore able to reduce disease burden compared to a different (lessresistant) plant (e.g., a different plant variety) grown in similardisease conditions. One of skill will appreciate that plant resistanceto disease conditions varies widely, and can represent a spectrum ofmore-resistant or less-resistant phenotypes. However, by simpleobservation, one of skill can generally determine the relativeresistance of different plants, plant varieties, or plant families underdisease conditions, and furthermore, will also recognize the phenotypicgradations of “resistant.”

One of skill will appreciate that plant resistance to disease conditionsvaries widely, and can represent a spectrum of more-resistant orless-resistant phenotypes. However, by simple observation, one of skillcan generally determine the relative resistance or susceptibility ofdifferent plants, plant lines or plant families under diseaseconditions, and furthermore, will also recognize the phenotypicgradations of “resistant.”

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

EXAMPLES Example 1 Identification of Recombinant Tomato Lines ComprisingTy-1

The Ty-1 TYLCV resistance locus from S. chilense LA1969 was introgressedinto elite S. lycopersicon plants, resulting in several breeding lineswith a high level of resistance to TYLCV. However, these lines exhibitedundesirable agronomic traits associated with the Ty-1 introgression fromS. chilense, including necrosis. The molecular markers shown in Table 1were developed for identification of recombinants maintaining TYLCVresistance with reduced incidence of necrosis.

TABLE 1 Markers on chromosome 6 for identification of Ty-1 recombinants.Marker Position NL0233580 24,527,065 NL0233570 3,293,791 NL023404522,036,414 NL0233833 34,148,452 NL0233541 3,294,160 NL0347537 28,852,768NL0347530 3,329,633 NL0347552 23,690,907 NL0244835 29,785,303 NL023118731,166,320 NL5113529 27,693,113 NL5113524 2,404,524 NL5113488 2,207,760NL5113492 634,983 NL5113500 2,842,081 NL5113496 3,540,443 NL511352030,622,375 NL5113518 1,162,062 NL5113489 1,238,761 NL5113515 21,357,322NL5113464 31,664,916 NL5132888 20,421,231 NL0215181 14,499,315 NL021601123,120,245 NL0217856 12,044,896 NL0216387 22,496,662 NL513290820,493,538 NL5132893 3,020,831 NL5132900 20,710,909 NL5132906 8,115,068

The use of these Ty-1 linked markers led to the identification of tomatolines 11039-1, 09GA0361-11 and 08TK01-26-2. As shown in FIG. 1, Line11039-1 comprises a recombination between markers Q_NL5113515 andQ_NL5113488. This line is TYLCV susceptible but still exhibits leafnecrosis, suggesting that the Ty-1 resistance locus is positioned to theright (south on the chromosome) of marker Q_NL5113515, while at leastsome component of the necrosis locus from the S. chilense introgressionis located to the left (north on the chromosome) of marker Q_NL5113488.Line 09GA0361-11 comprises a recombination to the right of CD67 and isTYLCV resistant, while exhibiting reduced leaf necrosis. An even smallerintrogression was identified in line 08TK01-26-2, which exhibits TYLCVresistance in the field and under more controlled conditions(agro-inoculation with TYLCV).

Example 2 Mapping Populations

Parent varieties Petit (FIR-191-PETIT) and Screen (FIR-191-SCREEN) werecrossed to create an F2 population for further analysis of necrosis andother traits in plants comprising Ty-1 introgressions. FIR-191-PETIT isthe parent comprising the chilense introgression. Ninety-two hundredseeds from the F2 population were sown and analyzed with markersNL0232070 and NL0231193 resulting in 209 validated recombinants. Ofthese recombinants, 42 were grown in the greenhouse to produce selfed(F3) seed, and F3 plants were phenotyped for necrosis. Twenty-five ofthe F3 lines fixed for a recombination event between NL0232070 andNL0231193 were grown in greenhouse conditions for analysis of the Ty-1introgression.

Example 3 Evaluation of TYLCV Resistance and Necrosis in MappingPopulations

Plants were evaluated for TYLCV resistance using the following whiteflyinoculation protocol. Plants were exposed to whiteflies (Bemisia tabaci(Q biotype)) carrying TYLCV for 48 hours at the first true leaf stage(approximately 3 weeks after sowing). Plants were scored for TYLCVresistance according to the scale shown in Table 2 and evaluated fornecrosis using the scale shown in Table 3. Scoring was performed 15 and30 days post inoculation when at least 90% of the susceptible controlplants were infected with TYLCV (score 9).

TABLE 2 TYLCV Scoring Scale Rating 1 = no symptoms. scale 3 = someyellow spots or stripes at the edges of top leaves. definition 5 =obvious yellowing of top leaves, no curling. 7 = obvious yellowing andcurling of top leaves. Some growth reduction of plant might occur. 9 =heavy growth reduction of plant, heavy curling and yellowing of topleaves. Norm to At least 90% of the susceptible control plants should bevalidate infected with TYLCV (score 9). bioassay

TABLE 3 Necrosis Scoring Scale. All leaves are left on the plant forscoring. Rating 1 = no necrosis; green leaf. scale 2 = leaf edge showingnecrosis; remaining leaf is still green. definition 3 = light necrosis;some clear necrotic spots on bottom leaves. No chlorosis. Green leaf. 4= light necrosis; light spots on the leaf and clear necrotic spots onleaf of bottom part of the plant. 5 = moderate necrosis; clear spots onleaf and clear necrotic spots on leaf of bottom part of the plant. 6 =necrosis and light chlorosis on leaf of bottom part of the plant. 7 =many necrotic spots and chlorosis on leaf of about 75% of the plant. 8 =severe necrosis and chlorosis. Only top of the plant has some greenleaves left. 9 = plant completely necrotic and chlorotic.

Example 4 Identification of Necrosis QTL

Seed of the F2 population described in Example 2 was sown and the plantswere grown under greenhouse conditions in winter without artificiallighting. Days were <12 h, night temperature was >15° C., and daytemperature was a maximum of <25° C. Necrosis was evaluated using theprotocol described in Example 3. F2 plants were sampled individually andgenotyped with a set of TaqMan assays of markers distributed across thegenome. Assay conditions were as follows: 20 sec at 95° C., followed by35 repeats of 3 sec at 95° C. and 20 seconds at 60° C., and holding at20° C.

Combining the genotypic and phenotypic data, two QTL with logarithm ofthe odds (LOD) values >3.0 could be detected using R/qtl with thedefault settings. The major QTL was located on chromosome 6, as shown inFIG. 2. Table 4 shows the LOD values for the markers flanking theidentified QTL on chromosomes 2 and 6.

TABLE 4 LOD data for markers showing significance for necrosis. MarkerChr. Pos. LOD NSLYC008374675 (SEQ ID NO: 5) 2 39 3.051654449NSLYC008375578 (SEQ ID NO: 6) 2 59.57 2.589825655 NL0250996 6 3.357.03813316 NSLYC009418370 (SEQ ID NO: 4) 6 6.94 49.53995957NSLYC008427230 6 17.31 36.44859245

Example 5 Mapping of Necrosis Loci within the Ty-1 Introgression onChromosome 6

The F3 populations described in Example 2 comprising S. chilense Ty-1introgressions were further analyzed to identify loci associated withnecrosis derived from Ty-1 introgressions. Table 5 shows necrosisscoring results for F3 populations, as well as average necrosis scoresfor each line at April and May time points. A set of markers wasdeveloped to genotype these F3 populations in the region surroundingTy-1, and these markers are shown in Table 6.

When analyzing the Ty-1 region with these markers, it was noted thatalmost no recombination was observed in the centromeric region ofchromosome 6, where Ty-1 is located. The development of additionalmarkers in the 5-25 cM region of chromosome 6 increased the possibilityfor detecting recombination events. These novel molecular markers alsomade it possible to screen large populations to select plants comprisingsmaller Ty-1 introgressions which exhibited high levels of resistance toTYLCV conferred by the Ty-1 resistance gene, while also reducing oreliminating the occurrence of undesirable agronomic traits previouslyassociated with Ty-1 introgressions, such as necrosis.

FIG. 3 shows genotyping data for control lines and F3 lines comprisingrecombination events between markers NL0232061 and NL231193. Averagenecrosis scores at the April and May time points are also shown. FIG. 4shows the average necrosis score for several of the plant lines shown inFIG. 3 based on the location of the break point between donor S.chilense DNA and S. esculentum DNA determined using the markers shown inFIG. 3. These data clearly demonstrate that plants comprising a Ty-1introgression wherein S. chilense Ty-1 donor DNA is replaced by S.esculentum DNA from the top of the chromosome until markerNSLYC008382908 do not exhibit the increased levels of necrosispreviously observed for plants with Ty-1 introgressions.

TABLE 5 Necrosis scoring results for F3 plants. April Avg May Avg F3Pop. No. Plant 1 Plant 2 Plant 3 Plant 4 Apr Plant 1 Plant 2 Plant 3Plant 4 May TJD0015 8 9 8 8 8 7 7 8 7 7.25 TJD0006 8 9 7 8 8 8 9 7 8 8TJD006¹ 2 2 1 3 2 1 2 1 2 1.5 TJD0013² 3 1 4 x 2.67 2 1 2 x 1.67 TJD00211 1 2 x 2.25 1 2 2 x 2.25 TJD0026 1 x x x 1 1 x x x 1 TJD0018 1 1 2 11.25 2 1 2 1 1.5 TJD0027 2 2 3 x 2.33 1 1 2 x 1.33 TJD0029 1 1 x x 1 2 2x x 2 TJD0020 1 1 1 x 1 1 1 1 x 1 TJD0017 2 2 1 1 1.5 1 1 1 1 1 TJD00076 5 2 4.33 5 4 2 3.67 TJD0022 8 8 9 8 8.25 7 7 9 7 7.5 TJD0014³ 7 1 x x4 4 1 x x 2.5 0014⁴ 7 8 5 x 6.67 6 7 3 x 5.33 0020⁴ 8 9 8 9 8.67 8 8 8 88 0021⁴ 8 8 8 9 8.25 8 8 8 9 8.25 TJD0028 3 9 8 9 7.25 2 8 7 9 6.5TJD0030 5 2 2 x 4.25 6 3 2 x 4.75 TJD0023 5 2 x x 3.5 6 2 x x 4 TJD00022 5 2 x 3 2 4 3 x 3 TJD0004 2 6 7 1 4 2 5 6 1 3.5 TJD0005 2 1 3 2 2 2 23 3 2.5 TJD0008 3 9 x x 6 3 7 x x 5 TJD0010 4 3 2 2 2.75 4 3 2 2 2.75TJD0009 2 1 2 3 2 2 1 2 1 1.5 0018⁴ 9 x x x 9 9 x x x 9 TJD0019 8 8 2 x6 7 7 3 x 5.67 ¹esc control ²ESC control ³one plant is heterozygous ⁴chicontrol

TABLE 6 Sequences associated with markers in Ty-1 region on tomatochromosome 6. Phys Gen. Marker Name Pos. Pos. SNP NL0232061 21,597,2264.32 G/A NL0216350 27,605,333 4.25 T/C NL0244835 29,785,303 7.47 GGTT/*NSLYC005134413 30,027,432 7.88 A/T NSLYC005134429 (SEQ ID NO: 3)30,199,948 8.23 */C NSLYC008382908 30,322,811 8.51 G/A NSLYC00837323230,523,713 8.93 C/T NSLYC009078368 30,623,862 9.14 C/A NSLYC00907796930,658,888 9.21 A/G NSLYC009077970 (SEQ ID NO: 2) 30,689,718 9.27 G/TNSLYC008383071 (SEQ ID NO: 1) 30,711,829 9.31 T/C NSLYC009418370 (SEQ IDNO: 4) 30,876,429 9.64 T/* NSLYC008426177 30,892,169 9.67 A/T NL023119331,800,043 11.6 A/C NL0232070 2,633,793 3.66 A/C NSLYC00513440529,150,417 6.4 C/G

Example 6 Identification of QTL Associated with Necrosis on Chromosome 2

As shown in FIG. 2 and Table 4, a QTL associated with necrosis wasidentified on chromosome 2. The TT_GG haplotype (the TT allele at markerNSLYC008374675 (SEQ ID NO:5), and the GG allele at marker NSLYC008375578(SEQ ID NO:6) is associated with reduced or absent necrosis as shown inTable 7 and FIG. 5. Necrosis in plants having a TT_GG haplotype istherefore associated with a different necrosis QTL. The CC_AA haplotype(the CC allele at marker NSLYC008374675 (SEQ ID NO:5), and the AA alleleat marker NSLYC008375578 (SEQ ID NO:6) is associated with necrosis.Tomato lines having this allele may exhibit necrosis, although typicallyat intermediate levels (FIG. 5). The VIC probe sequences, FAM probesequences, and forward and reverse primers for marker NSLYC008374675 andmarker NSLYC008375578 are provided as SEQ ID NOs:7-8, 9-10, 11-12, and13-14, respectively.

TABLE 7 Markers associated with necrosis on tomato chromosome 2. SEQPhys Non-necrotic Necrosis Marker Name ID No. Pos. allele alleleNSLYC008374675 5 34,929,994 TT CC NSLYC008375578 6 38,823,651 GG AA

Example 7 TaqMan Assay

A TaqMan assay was developed to detect a SNP on chromosome 6 (baseposition 32,987,053) using marker NSLYC008426102. Genotyping results ona set of F4 recombinant lines with TaqMan assays located in the Ty-1region between 20 and 34 Mbp of chromosome 6. The TaqMan assay showed100% correlation on the existing recombinant population (FIG. 6).

Example 8 Trait Introgression of TYLCV Resistance without Necrosis

A tomato line comprising a conventional Ty-1 introgression andexhibiting necrosis is crossed with a tomato line comprising the reducedTy-1 introgression described herein and exhibiting little or nonecrosis. Progeny plants from this cross are backcrossed to produce aBC1 generation. BC1 progeny plants are selected for the S. esculentumallele at marker NSLYC009077970 (SEQ ID NO:2) indicating the presence ofthe reduced Ty-1 introgression of the invention. BC1 progeny plants orfurther progeny plants are selected for the presence of the S. chilenseallele at marker NSLYC009418370 (SEQ ID NO:4) to follow the Ty-1introgression. Additionally, the incidence of necrosis may be furthermodulated by selecting for alleles associated with an absence ofnecrosis at the described chromosome 2 QTL interval by using markersNSLYC008374675 (SEQ ID NO:5) and NSLYC008375578 (SEQ ID NO:6).

Example 9 Recombinant Tomato Plants with a Smaller Ty-1 IntrogressionSize

To create tomato plants with a Ty-1 introgression having less donor S.chilense DNA, initial selection for recombination between markersNSLYC008382908 (8.51 cM) and NSLYC008383071 (9.31 cM, SEQ ID NO:1) wasperformed in order to break linkage between Ty-1 and undesirablenecrosis drag (see FIG. 7). Next, selection for recombination betweenmarker NSLYC009418370 (9.64 cM, SEQ ID NO:4) and NL231193 (11.58 cM) tominimize the amount of S. chilense DNA and thus reduce the size of theintrogression. Sequences for markers NSLYC008382908 and NL231193 areprovided in Table 9. Select recombinants were tested for resistance toTYLCV by inoculation with whiteflies on 4 replicates of 10 plants each.Additionally, whole plant harvesting of mature fruit and total harvestweight and fruit count were performed on 4 replicates of 6 plants each.The results of the performance testing are shown in Table 8 and FIGS. 8and 9.

TABLE 8 Mean values for one-way ANOVA of efficacy testing. Student'sGroup Group t test Background A B Set DI LSM (α = 0.05) FDR-14-818 P RPTY1-1 9.00 A FDR-15-2061 RC RP TY1-2 9.00 A FDR-15-2061 P RP TY1-3 9.00A FDR-15-2061 P RP TY1-4 9.00 A FDR-15-2061 RC RP TY1-4 9.00 AFDR-15-2061 P RP TY1-5 8.90 A FDR-15-2061 RC RP TY1-5 8.90 A FDR-15-2061P RP TY1-2 8.70 A FDR-14-818 RC RP TY1-1 8.40 A FDR-15-2061 RC RP TY1-37.48 B FDR-15-2061 RC D TY1-5 4.65 C FDR-15-2061 RC D TY1-4 4.20 C, DFDR-15-2061 RC D TY1-2 4.18 C, D FDR-9Q08133 P D TY1-1 3.80 D, EFDR-15-2061 RC D TY1-3 3.65 D, E, F FDR-15-2107 P D TY1-5 3.25 E, FFDR-15-2107 P D TY1-4 3.04 E, F FDR-15-2107 P D TY1-3 2.97 F FDR-15-2107P D TY1-2 2.89 F FDR-14-818 RC D TY1-1 2.03 G

Sequence capture was performed in order to characterize theintrogression on tomato chromosome 6 (FIG. 10). The introgression wasdetermined to be approximately 260 kb, and plants into which thisintrogression was inserted retained resistance to TYLCV while exhibitinga lack of necrosis, and fruit size and fruit set drag.

TABLE 9 Marker Sequences. Al- VIC FAM F R Marker leles Sequence SequenceSequence Sequence NSLYC G/A CTCAATGG CCTCAATG AGAAAAAT GGTAGCCTT 00838CAAACAA ACAAACAA GTGGCCAT AGATGCAAT 2908 (SEQ ID (SEQ ID GGGTAACTAGTGTGA NO: 15) NO: 16) (SEQ ID (SEQ ID NO: 17) NO: 18) NL023 A/CTCTACACAA TCTACACAA CTTGGGAG GCCCAACAG 1193 AAGAATGC ACGAATGC ATACTCTCATGATCTTT (SEQ ID (SEQ ID TGTTGCTT AAGAATGG NO: 19) NO: 20) (SEQ ID(SEQ ID NO: 21) NO: 22)

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A tomato plant of a cultivated tomato plantvariety comprising a recombinant introgression from Solanum chilense onchromosome 6, wherein said recombinant introgression comprises a firstallele conferring improved resistance to tomato yellow leaf curl virusrelative to a plant lacking said first allele, and wherein saidrecombinant introgression lacks a second allele genetically linked tosaid first allele and conferring necrosis.
 2. The tomato plant of claim1, wherein said recombinant introgression from Solanum chilense islocated between approximately 30.20 Mbp and 30.88 Mbp on chromosome 6.3. The tomato plant of claim 2, wherein said recombinant introgressionfrom Solanum chilense is located between approximately 30.69 Mbp and30.88 Mbp on chromosome
 6. 4. The tomato plant of claim 3, wherein saidrecombinant introgression from Solanum chilense is located betweenapproximately 30.71 Mbp and 30.88 Mbp on chromosome
 6. 5. The tomatoplant of claim 1, wherein said recombinant introgression from Solanumchilense is located between approximately 30.32 Mbp and 30.71 Mbp onchromosome
 6. 6. The tomato plant of claim 1, wherein said recombinantintrogression from Solanum chilense is located between approximately30.88 Mbp and 31.80 Mbp on chromosome
 6. 7. The tomato plant of claim 1,wherein the plant comprises a Solanum chilense allele at locusNSLYC008383071 (SEQ ID NO:1) and lacks a Solanum chilense allele atlocus NSLYC005134429 (SEQ ID NO:3).
 8. The tomato plant of claim 7,wherein the plant comprises a Solanum chilense allele at locusNSLYC008383071 (SEQ ID NO:1) and lacks a Solanum chilense allele atlocus NSLYC009077970 (SEQ ID NO:2).
 9. The tomato plant of claim 1,wherein the plant comprises S. chilense donor DNA within a genomicsegment flanked by NSLYC005134429 (SEQ ID NO:3) and NSLYC008383071 (SEQID NO:1).
 10. The tomato plant of claim 9, wherein the plant comprisesS. chilense donor DNA within a genomic segment flanked by NSLYC009077970(SEQ ID NO:2) and NSLYC008383071 (SEQ ID NO:1).
 11. The tomato plant ofclaim 1, wherein the plant comprises S. chilense donor DNA within agenomic segment flanked by NSLYC008382908 and NSLYC008383071 (SEQ IDNO:1).
 12. The tomato plant of claim 1, wherein the plant comprises S.chilense donor DNA within a genomic segment flanked by NSLYC009418370(SEQ ID NO:4) and NL231193.
 13. A plant part of the plant of claim 1.14. The plant part of claim 13, wherein the plant part is a cell, aseed, a root, a stem, a leaf, a fruit, a flower, or pollen.
 15. A methodfor producing a tomato plant with improved resistance to tomato yellowleaf curl virus comprising: (a) crossing the tomato plant of claim 1with itself or with a second tomato plant of a different genotype toproduce one or more progeny plants; and (b) selecting a progeny plantcomprising said recombinant introgression.
 16. The method of claim 15,wherein selecting the progeny plant comprises identifying a progenyplant that (1) comprises a Solanum chilense allele at a locusgenetically linked to said first allele and/or lacks an allele presentat the corresponding locus in the tomato plant, and (2) lacks a Solanumchilense allele at a locus genetically linked to said second allele thatconfers necrosis, and/or comprises an allele present at thecorresponding locus from in the tomato plant.
 17. The method of claim16, wherein selecting said progeny plant comprises marker-assistedselection (MAS).
 18. The method of claim 17, wherein marker-assistedselection (MAS) comprises detecting at least one allele at a locusselected from the group consisting of NSLYC005134429 (SEQ ID NO:3),NSLYC009077970 (SEQ ID NO:2), NSLYC009418370 (SEQ ID NO:4),NSLYC008383071 (SEQ ID NO:1), NSLYC008382908, and NL231193.
 19. Themethod of claim 15, wherein the progeny plant is an F2-F6 progeny plant.20. The method of claim 15, wherein producing the progeny plantcomprises backcrossing.
 21. The method of claim 20, wherein backcrossingcomprises from 2-7 generations of backcrossing.
 22. A method forobtaining a tomato plant exhibiting improved resistance to tomato yellowleaf curl virus comprising: (a) obtaining a tomato plant heterozygousfor a first allele that confers resistance to tomato yellow leaf curlvirus and that is genetically linked in the plant to a second allelefrom Solanum chilense that confers necrosis; (b) obtaining progeny ofthe plant; and (c) selecting at least a first progeny plant in whichrecombination has occurred such that the progeny comprises said firstallele that confers resistance to tomato yellow leaf curl virus but notsaid second allele that confers necrosis; wherein selecting said firstprogeny plant comprises detecting at least one allele at a locusselected from the group consisting of NSLYC005134429 (SEQ ID NO:3),NSLYC009077970 (SEQ ID NO:2), NSLYC009418370 (SEQ ID NO:4),NSLYC008383071 (SEQ ID NO:1), NSLYC008382908, and NL231193.
 23. Themethod of claim 22, wherein the progeny plant is an F2-F6 progeny plant.24. The method of claim 22, wherein producing the progeny plantcomprises backcrossing.
 25. The method of claim 24, wherein backcrossingcomprises from 2-7 generations of backcrossing.
 26. A plant produced bythe method of claim
 22. 27. A part of the plant of claim 26, selectedfrom the group consisting of a cell, a seed, a root, a stem, a leaf, afruit, a flower, and pollen.
 28. A tomato plant of a cultivated tomatoplant variety comprising a TT allele at marker NSLYC008374675 (SEQ IDNO:5), and a GG allele at marker NSLYC008375578 (SEQ ID NO:6), whereinsaid alleles confer a lack of or resistance to necrosis.
 29. The methodof claim 22, further comprising: (d) selecting against alleles onchromosome 2 associated with necrosis; wherein said selecting againstcomprises selecting against plants comprising a CC allele at markerNSLYC008374675 (SEQ ID NO:5), or an AA allele at marker NSLYC008375578(SEQ ID NO:6), wherein the presence of said alleles confers necrosis.30. A method for obtaining a tomato plant exhibiting resistance tonecrosis comprising: (a) obtaining a tomato plant heterozygous for afirst allele that confers resistance to necrosis; (b) obtaining progenyof the plant; and (c) selecting at least a first progeny plant in whichrecombination has occurred such that the progeny comprises said firstallele that confers resistance to necrosis; wherein selecting said firstprogeny plant comprises detecting at least one allele at a locusselected from the group consisting of marker NSLYC008374675 (SEQ IDNO:5) and marker NSLYC008375578 (SEQ ID NO:6), wherein said allelesconfer a lack of or resistance to necrosis.
 31. A method for obtaining atomato plant exhibiting improved resistance to tomato yellow leaf curlvirus comprising: (a) obtaining a tomato plant heterozygous for a firstallele from Solanum chilense that confers resistance to tomato yellowleaf curl virus and that is genetically linked in the plant to a secondallele from Solanum chilense that confers necrosis; (b) obtainingprogeny of the plant; and (c) selecting at least a first progeny plantin which recombination has occurred such that the progeny comprises saidfirst allele that confers resistance to tomato yellow leaf curl virusbut not said second allele that confers necrosis; wherein selecting saidfirst progeny plant comprises selecting a progeny wherein recombinationhas occurred between locus NSLYC009418370 (SEQ ID NO:4) and locusNL231193.
 32. The method of claim 31, wherein said selecting comprisesdetecting at least one allele at a locus selected from the groupconsisting of NSLYC008383071 (SEQ ID NO:1), NSLYC008382908,NSLYC009418370 (SEQ ID NO:4), and NL231193.
 33. The method of claim 31,further comprising: (d) selecting a further progeny plant from among theplants selected in step (c) or subsequent progeny thereof in which afurther recombination has occurred between locus NSLYC009418370 (SEQ IDNO:4) and locus NL231193.
 34. The method of claim 31, wherein selectingcomprises identifying the progeny plant as lacking one or more traitselected from of necrosis, reduced fruit set, and reduced fruit sizerelative to a plant in which the recombination has not occurred.
 35. Aplant produced by the method of claim 31.